2-Butanone, 1,1,1,3,4,4,4-heptafluoro-3-(trifluoromethyl)-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 June - 8 June 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test substance is reactive with water, but neither the parent material nor the potential degradation products other than trifluoroacetic acid were detected in the test solutions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,1,1,3,4,4,4-heptafluoro-3-(trifluoromethyl)butan-2-one

EC Number:

690-995-3

Cas Number:

756-12-7

Molecular formula:

C5 F10 O

IUPAC Name:

1,1,1,3,4,4,4-heptafluoro-3-(trifluoromethyl)butan-2-one

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report: MTDID 20422
- Substance type: Pure active substance
- Physical state: Volatile liquid (VP 87.5 kPa at 20 °C)
- Analytical purity: >99%
- Expiration date of the lot/batch: 16 Feb 2013
- Stability under test conditions: Reactive to water
- Storage condition of test material: Sealed container

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: 2 or 4 replicates of each group were taken for sampling (volume: 3 mL) at times 0h, 24h, 48h, and 72h. Samples at t = 0h were taken from excess prepared solution. Samples at 24h and 48h were taken from additional test vessels set aside for sample collection. Samples from 72h were taken from the test solutions. Duplicate samples were taken and analyzed at 24 hours.
- Sample storage conditions before analysis: Not applicable, samples were transported on dry ice to the test site and analysed on the day of sampling. Samples were analysed starting at 1.5 hours after arrival at the laboratory.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A volume of 64 µL CAS# 756-12-7 corresponding to 100 mg/L was injected into 1 L of test medium and magnetically stirred for 17 min, after which no test substance was visible in the medium. The flask was closed during stirring, but a small amount of headspace was present. Lower test concentrations were prepared by susbsequent dilutions of the highest concentration in test medium.
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None, final test solutions were all clear and colorless.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): Three or four days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E+04 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
ACCLIMATION
- Acclimation period: None
- Culturing media and conditions (same as test or not): Pre-culture was maintained under the same conditions as used in the test.
- Any deformed or abnormal cells observed: not reported

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

22.2 - 23.2 °C

pH:

7.2 - 10

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10, 32, and 100 mg/L (CAS# 756-12-7)
Measured: 1, 2, 4, 10, and 31 mg/L (Degradation product Trifluoro acetic acid)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass, air-tight closed, containing 75 ml of test solution (headspace reduced to minimum)
- Agitation: Yes, during incubation the algal cells were kept in suspension by continuous shaking.
- Control end cells density: 64.9E+04 cells/ml
- No. of colonies per vessel: Initial cell density was 1E+04 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
1 replicate of the highest concentrations without algae
2 or 4 replicated of each group for sampling purposes

GROWTH MEDIUM
- Standard medium used: yes/no: Yes - M2 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water used to make growth medium was purified by reverse osmosis.
- Culture medium different from test medium: yes, propagation in M1, test in adjusted M2 (NaHCO3 increased to 100 mg/L to compensate for sealed bottle test).
- Intervals of water quality measurement: pH at beginning, 24, 48, and 72 hours, temperature continuously

OTHER TEST CONDITIONS
- Adjustment of pH: no, the pH in a number of groups exceeded 9.0 at the end of the test due to accumulation of CO2 produced during exposure. Exponential growth was observed during the entire period of exposure in the control vessel and the validity criteria were met. Therefore, it was assumed that this observation had no influence on the results of this study.
- Photoperiod: continuous illumination using 30 Watt 'Cool-white' TLD-Iamps. Light intensity within the range of 99 to 106 µE/(m²∙s)
-Other: Algae were introduced within 66 minutes after preparation of the initial stock solution

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Other: Cell counts at 0h, 24h, 48h, and 72h were determined by spectrophotometric measurement at 720 nm using a Shimadzu Spectrophotometer UV-1800 with immersion probe (UVProbe 2.33 software: Algal cell density) (pathlength =20 mm). Algal medium was used as blank.
- Cells were observed microscopically after the end of the 72-h exposure period in the two highest concentrations to check for any abnormal appearance of the algae.

RANGE-FINDING STUDY
- Test concentrations: Control and a range of 0.1 to 100 mg/l (nominal concentrations of CAS# 756-12-7) increasing by a factor of 10
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

98 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % C.I. 67.7 - 140 mg/L. Concentration is stoicheometric equivalent of measured TFA concentration.

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.3 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Concentration is stoicheometric equivalent of measured TFA concentration.

Details on results:

- Exponential growth in the control (for algal test): yes/no: Yes (Figure 1)
- Growth in controls (validity criteria):
- cell density increase to hour 48: factor of >16
- % Coefficient of variance in average specific growth rate at hour 72: 2% (Table 1)
- % Coefficient of variance in section-specific growth rates to hour 72: 22% (Table 2)

- Any visual signs of phytotoxicity (abnormalities): Observation was done at the end of the test and no abnormalities were found.
- Any stimulation of growth found in any treatment: No Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: CAS#756-12-7 is reactive with water. The concentrations of CAS# 756-12-7, as well as the potential degradation products were measured. The concentrations of CAS# 756-12-7 and all degradation products except trifluoroacetic acid were below the limit of detection of the analytical method, i.e. 1 mg/L. The measured concentrations of the degradation product, trifluoroacetic acid remained stable during the entire test period (75-100% of initial) (Table 3). The effect parameters were calculated in terms of initially measured concentrations of trifluoro acetic acid. Effects concentrations were then expressed in terms of stoicheometric equivalent concentration of CASVAF#

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.98 mg/L, 95% C.I. 0.74 - 1.3 mg/L. The historical ranges for growth rate reduction for Pseudokirchneriella subcapitata lie between 0.82 and 2.3 mg/l. Hence, the growth rate reduction EC50: 72h for the Pseudokirchneriella subcapitata tested corresponds with this range.

Reported statistics and error estimates:

ANOVA, Bonferroni t-test and TOXSTAT Release 3.5 were utilized to evaluate statistically significant differences from the controls. Calculation of the EC50 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the initially measured concentrations of trifluoro acetic acid.

Any other information on results incl. tables

Table 1: Cell Density, Yield Inhibition and Growth Rate Reductions
Measured Concentration, Trifluoroacetic acid, mg/L (nominal concentration, CAS# 756-12-7, mg/L)	Vessel Number	Individual Cell Densities, 10^4 cells/mL				Yield	Yield Inhibition (%)	Growth Rate (µ)	Growth Rate Reduction (%)
		Exposure Period (hours)
		0	24	48	72	0 - 72 h	0 - 72 h	0 - 72 h	0 - 72 h
Control	1	1.00	4.95	23.13	61.84	60.84		0.05728
	2	1.00	3.95	21.22	54.43	53.43		0.05551
	3	1.00	4.21	24.79	74.00	73.00		0.05978
	4	1.00	4.06	24.65	65.36	64.36		0.05805
	5	1.00	3.86	23.15	65.18	64.18		0.05802
	6	1.00	3.67	21.97	68.49	67.49		0.05870
	Mean					63.88		0.05789
								CV, 2%
1 (1)	1	1.00	3.68	20.32	58.96	57.96	9	0.05662	2
	2	1.00	3.38	17.75	54.55	53.55	16	0.05554	4
	3	1.00	2.60	21.07	56.94	55.94	12	0.05614	3
	Mean					55.81	12.6	0.05610	3.1
2 (3.2)	1	1.00	1.93	14.76	44.94	43.94	31	0.05285	9
	2	1.00	3.25	18.99	57.32	56.32	12	0.05623	3
	3	1.00	3.20	16.62	52.46	51.46	19	0.05500	5
	Mean					50.57	20.8	0.05469	5.5
4 (10)	1	1.00	2.51	12.27	34.90	33.90	47	0.04934	15
	2	1.00	2.53	11.59	29.34	28.34	56	0.04693	19
	3	1.00	2.60	10.98	33.79	32.79	49	0.04889	16
	Mean					31.68	50.4	0.04839	16.4
10 (32)	1	1.00	2.07	8.56	18.10	17.10	73	0.04022	31
	2	1.00	2.10	8.49	16.85	15.85	75	0.03922	32
	3	1.00	1.80	9.28	19.12	18.12	72	0.04098	29
	Mean					17.02	73.4	0.04014	30.7
31 (100)	1	1.00	2.67	7.05	9.80	8.80	86	0.03170	45
	2	1.00	2.14	6.69	9.44	8.44	87	0.03118	46
	3	1.00	2.33	7.36	10.51	9.51	85	0.03267	44
	Mean					8.92	86.0	0.03185	45.0

Table 2: Section-by-section Growth Rates
Measured concentration, Trifluoroacetic acid, mg/L (nominal concentration, CAS# 756-13-8, mg/L)	Vessel Number	Growth Rate (µ)			Growth Rate Reduction (%)
		Exposure Period (hours)			Exposure Period (hours)
		0 - 24h	24-48h	48-72h	0 - 24h	24-48h	48-72h
Control	1	0.06664	0.06425	0.04097
	2	0.05720	0.07010	0.03925
	3	0.05986	0.07391	0.04556
	4	0.05837	0.07516	0.04063
	5	0.05625	0.07466	0.04314
	6	0.05414	0.07460	0.04738
	Mean	0.05874	0.07211	0.04282
	CV (%)	7	6	7
	The mean CV for section-by-section specific growth rate was: 22%
1 (1)	1	0.05427	0.07122	0.04438	8	1	-4
	2	0.05076	0.06909	0.04678	14	4	-9
	3	0.03986	0.08712	0.04143	32	-21	3
	Mean	0.04829	0.07581	0.04419	17.8	-5.1	-3.2
2 (3.2)	1	0.02742	0.08475	0.04639	53	-18	-8
	2	0.04905	0.07361	0.04604	17	-2	-8
	3	0.04850	0.06861	0.04789	17	5	-12
	Mean	0.04166	0.07565	0.04677	29.1	-4.9	-9.2
4 (10)	1	0.03841	0.06606	0.04355	35	8	-2
	2	0.03871	0.06339	0.03868	34	12	10
	3	0.03976	0.06006	0.04685	32	17	-9
	Mean	0.03896	0.06317	0.04303	33.7	12.4	-0.5
10 (32)	1	0.03039	0.05908	0.03117	48	18	27
	2	0.03099	0.05814	0.02854	47	19	33
	3	0.02451	0.06830	0.03013	58	5	30
	Mean	0.02863	0.06184	0.02995	51.3	14.2	30.1
31 (100)	1	0.04098	0.04042	0.01370	30	44	68
	2	0.03168	0.04752	0.01433	46	34	67
	3	0.03523	0.04793	0.01487	40	34	65
	Mean	0.03596	0.04529	0.01430	38.8	37.2	66.6

 

Table 3. Measured concentrations in the final test
Time of sampling (hours)	Nominal concentration, CAS# 756-12-7 (mg/L)	Analysed concentration, Trifluoroacetic acid (mg/l)	Relative to initial (%)
0	control	< LOD ¹	─
	1	1
	3.2	2
	10	4
	32	10
	100	31
	100 WA ¹	31
24	Control	< LOD
	Control	< LOD	─
	1.0	1
	1.0	1 (1) ²	100
	3.2	2
	3.2	2 (2)	100
	10	3
	10	3 (3)	75
	32	10
	32	10 (10)	100
	100	31
	100	29 (30)	97
	100 WA	31
	100 WA	32 (32)	102
48	Control	< LOD	─
	1.0	1	100
	3.2	2	100
	10	3	75
	32	11	110
	100	31	100
	100 WA	30	97
72	Control	< LOD	─
	1.0	1	100
	3.2	2	100
	10	4	100
	32	10	100
	100	31	100
	100 WA	31	100
1, LOD – limit of detection (LOD=1 mg/l); WA – without algae 2, () – average concentration calculated on the basis of two measurements.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour EC50 of CAS# 756-12-7 to Pseudokirchneriella subcapitata is 98 mg/L. The 72-hour NOEC of CAS# 756-12-7 to Pseudokirchneriella subcapitata is 2.3 mg/L.

Executive summary:

The toxicity of CAS# 756-12-7 to the green algae, Pseudokirchneriella subcapitata, was assessed in a 72-hour toxicity test conducted according to the OECD 201 (2011) method. Test containers were sealed to avoid volatilization losses of test material, and growth medium was amended with 100 mg/L NaHCO3 to provide sufficient carbon dioxide and buffering to maintain exponential growth. An exponential growth was observed during the entire period of exposure in the control vessel and the validity criteria were met.

CAS# 756-12-7 is reactive with water. The concentrations of parent, as well as the potential degradation products were measured. The concentrations of CAS# 756-12-7 and all degradation products except trifluoroacetic acid were below the limit of detection of the analytical method, i.e. 1 mg/L. The measured concentrations of the degradation product, trifluoroacetic acid (TFA), remained fairly stable during the entire test period (75-100% of initial). Based on the obtained results, the effect parameters were expressed in terms of initially measured concentrations of TFA. The measured concentrations for the degradation product, TFA were: 1, 2, 4, 10, and 31 mg/L. Effect concentrations for the parent were calculated from TFA using the molecular weight ratio on a stoicheometric basis.

The 72-hour ErC50 of CAS# 756-12-7 to Pseudokirchneriella subcapitata was 98 mg/L based on growth rate. Neither the parent material nor other degradation products were detected in the test solutions.

The study was performed in accordance with internationally-accepted test guidelines and Good Laboratory Practice (GLP) standards. However, neither the parent material nor degradation products, other than TFA, were detected in the test solutions. Therefore, this study is reliable with restrictions and the results are suitable for purposes of Risk Assessment, Classification & Labeling, and PBT Analysis.

Results Synopsis

Test Type: static (based on data obtained using OECD201 methodology)

EC50: 98 mg/L 95% C.I.: 67.7 to 140 mg/L

NOEC: 2.3 mg/L